Hepatitis C virus (HCV) is the main pathogen causing chronic hepatitis, liver cirrhosis and, in some instances, hepatocellular carcinoma [refer to reference 14]. Although HCV affects more than 3% of the world population, no specific and efficient anti-HCV therapy has yet been developed.
HCV contains a single, positive-stranded RNA genome of about 9,600 nucleotides in length encoding a polyprotein of about 3010 amino acids [Reference 6]. This polyprotein precursor is co- or post-translationally processed into at least 10 mature structural and nonstructural proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) by cellular and viral proteases [References 6 and 23].
HCV NS5B harbors RNA-dependent RNA polymerase activity [Reference 2], which is considered crucial for the synthesis of negative-strand and genomic viral RNA during HCV genome replication. Therefore, HCV NS5B is believed to be essential for viral proliferation, and hence, is a primary target for the development of antiviral drugs [Reference 18].
Characteristics of RNAs, in that they can adopt complex but stable structures to specifically and readily bind to target proteins, and can be chemically synthesized with ease, make RNAs potentially very useful diagnostic and/or therapeutic leading compounds [References 4 and 8].
Short RNA ligands, termed RNA aptamers, have been identified from a random RNA library to bind to a wide variety of proteins with high affinity and specificity using in vitro iterative selection techniques, called Systemic Evolution of Ligands by Exponential enrichment (SELEX) [References 7 and 28].
Several aptamers have been successfully evaluated in animal disease models [References 9, 24 and 26], and some of them are now in the therapeutic clinical development stage [Reference 27]. Of note, the U.S. FDA recently approved an RNA aptamer against anti-vascular endothelial growth factor (VEGF), called pegaptanib sodium (Macugen), for the treatment of all types of neovascular age-related macular degeneration [Reference 19], signifying tremendous therapeutic potential of RNA aptamers.
The isolation and characterization of high-affinity RNA aptamers specific for HCV NS5B has recently been achieved [References 3 and 29]. Although the isolated aptamers have been shown to inhibit the enzymatic activity of RNA-dependent RNA polymerase in vitro, no studies have described the inhibition of intracellular HCV replication with RNA aptamers against HCV NS5B.
Leading to the present invention, intensive and thorough research into inhibition against intracellular HCV replication, conducted by the present inventors, resulted in the finding that RNase-resistant RNA aptamers for HCV NS5B RNA-dependent RNA polymerase can inhibit HCV replication in human hepatoma cell lines.